The heat shock proteins (HSP) represent a ubiquitous stress response; they are found in all organisms and cell types. Work in the Principal Investigator's laboratory focuses on HSP 72, one of the major heat shock proteins. Loss of HSP 72 is a lethal mutation. HSP 72 increases in response to stress; in the heart it increases in response to ischemia. Work in the Principal Investigator's laboratory is focused on understanding the molecular mechanisms of cellular injury that underlie ischemic damage in the heart. When HSP 72 is expressed in response to stress it translocates to the nucleus, not only the heart, but in all other stress responses that have been studied in different cell and tissue types. The mechanism of this movement to the nucleus and the function of HSP 72 in the nucleus are unknown. This proposal focuses on the regulation of nuclear translocation of HSP 72 with stress. In preliminary experiments with Cos cells, a common model for nuclear localization studies, it was determined that a change in phosphorylation at tyrosine 524 (Y524) occurs prior to nuclear localization. This proposal will follow-up on this initial observation. The investigator's hypothesize that nuclear localization of HSP 72 is an essential part of its protective functions, and that phosphorylation of tyrosine to phenylalanine, a conservative mutation preserving the aromatic side chain, and to aspartic acid, which mimics phosphorylation by placing a permanent negative charge at this site. These two mutants will be studied for nuclear localization with heat stress using the Cos cell model, already well-established in the laboratory. Additional work will be done with tyrosine kinase inhibitors to try to inhibit nuclear localization of HSP 72 with heat shock. Specific Aim 2 focuses on identification of the kinase that phosphorylates Y524. The yeast two-hybrid system will be used for this work. Specific Aim 3 will examine phosphorylation of HSp 72 in neonatal cardiocytes with hypoxia and reoxygenation to determine whether HSP 72 is phosphorylated with hypoxia or reoxygenation. The Y524 phosphorylation site is highly conserved with the same tyrosine phosphorylation site (with surrounding sequence) present in as diverse organisms as maize, drosophila, and man. Thus, the investigators postulate that phosphorylation with stress is conserved as well as sequence in this highly conserved, essential protein. The mutants, created in Specific Aim 1, will be expressed in the neonatal cardiocytes and the effect of modification of nuclear localization on myocyte viability with hypoxia and reoxygenation determined. The planned work will increase overall understanding of HSP 72 and the regulation of the stress response, and will contribute to the understanding of the response of the myocardium to hypoxia and reoxygenation. These studies will further the ultimate goal of understanding the regulation and function of this endogenous protective protein so that expression can be manipulated for the benefit of cardiac patients.